Despite intensive research efforts over the past 50 years, Staphylococcus aureus remains a serious threat to human health. In fact, recent reports of isolates with reduced susceptibility to vancomycin are indicative of the growing likelihood that S. aureus may become resistant to every currently available antimicrobial agent. Therefore, S. aureus represents a bigger threat to human health now than at any time since the pre-antibiotic area. The long-term goal of the investigators' research is to develop novel anti-staphylococcal therapeutic agents to stem the advance of drug-resistant strains. The current proposal is focused on the interaction of two globally-acting regulators of virulence gene expression: sar and agr. Previous work by the investigators and others indicate that the product of the sar gene, SarA, is a transcription factor that activates agr expression and leads to enhanced virulence of the organism. Because agr, sar and agr/sar mutants are less virulent in animal models of staphylococcal disease, they believe that the SarA/agr interaction is a very promising target for novel inhibitors of staphylococcal virulence. Importantly, since sar and agr mutants grow normally, but are less virulent, there should be reduced evolutionary pressure to become resistant to this type of inhibitor in comparison with classical antibiotics that inhibit growth. In addition, there are reports of increased sensitivity of sar and agr mutants to existing antimicrobial agents including methicillin. Inhibitors of the SarA/agr interaction may therefore be useful with respect to increasing the utility of existing drugs. To provide the foundation for inhibitor development, they will first reveal the mechanism by which SarA activates agr gene expression. The first three Specific Aims of the proposed research are: 1) to define the cis regulatory elements responsible for activation of the agr genes by SarA, 2) to define the functional domains of SarA required for activation of agr transcription, 3) to define the interaction of SarA with the agr regulatory elements at the atomic level. These aims will be accomplished by a combination of mutagenesis, in vitro and in vivo activity assays, x-ray crystallography, and atomic force microscopy. The fourth Specific Aim, to begin to develop anti-staphylococcal inhibitors that target these global regulatory factors, is intended to test the hypothesis that SarA-agr is a suitable target for novel anti-staphylococcal drugs. This broad-based approach takes advantage of a coordinated team, each member of which is an expert in his/her field.